Epithelial Stem Cell Programs

描述(由申请人提供)：成人干细胞具有巨大的固有的增殖能力，但又避免了与肿瘤生长相关的不朽。控制干细胞这种“自我更新”的遗传途径尚不清楚，但造血干细胞和精原干细胞是这类项目的关键“主要调节者”。一个相应的复层上皮，包括表皮、乳腺和前列腺的主调节器仍然难以捉摸，暴露了我们对这些高度再生和易患癌症的组织的理解的一个重大差距。然而，最近的数据确定这个缺失的上皮干细胞调控因子是P63，它是P53转录因子家族的成员。令人不安的是，全基因组芯片数据表明，p63直接调控着800多个基因，这暗示着干细胞自我更新的遗传程序可能很复杂，而且难以分析。在这个提议中，我们提出了这样的假设，即自我更新是由不同谱系的干细胞中的共同机制控制的，尽管已知的自我更新的主要调节因子是组织特异性的，这一矛盾迫在眉睫。我们假设的关键是，这些主调节者必须协调三个遗传程序来维持干细胞，其中两个决定了它们的组织特异性。因此，除了控制自我更新计划外，这些主调控者还需要推动与干细胞利基的互动，以及抢占组织特异性分化计划的先机。为了确定潜在的自我更新的共同遗传程序，我们在四个目标上建立了实验策略，这些策略利用了上皮干细胞独特的克隆形成特性。在目标1中，我们通过“屏蔽”p63的组织特异性作用来识别与自我更新有关的基因。为了做到这一点，我们比较了表型相似的相同谱系的上皮干细胞的基因表达谱，这些干细胞只是在增殖史上不同。在目标2中，我们确定了在造血细胞中受Bmi-1直接调控的基因，并将这些基因与上皮干细胞中受p63调控的基因进行比较，以寻找构成共同自我更新程序的相互调控的基因亚群。在目标3中，我们通过筛选受p63调控的转录因子子集的shRNA来解决这个问题，以确定它们在复制时间内的表达。我们的目标是使用来自目标1、目标2和目标3的所有三个数据集，为个体生成一个有限的候选基因列表，深入分析自我更新途径中的功能。在目标4中，我们研究了控制P63稳定性的分子机制，因为这一特征最终控制着自我更新。我们预计，这些策略将为p63和其他组织特异性的主调控因子向再生细胞传递干细胞的假定的“共同”途径提供基本的见解。 公共卫生相关性：对这里提出的问题的回答将揭示对成人干细胞，特别是上皮干细胞的治疗应用至关重要的信息。鉴于上皮组织对大多数人类癌症的贡献，这项工作也应该有助于揭示依赖p63的再生特性如何在肿瘤发生过程中做出贡献。

DESCRIPTION (provided by applicant): Adult stem cells possess an immense, intrinsic capacity for proliferation and yet avoid the immortality associated with neoplastic growth. The genetic pathways that control this "self-renewal" in stem cells are unknown, but key, "master regulators" of such programs have been described for hematopoietic and spermatogonial stem cells. A corresponding master regulator of stratified epithelia, including epidermis, breast, and prostate, had remained elusive, exposing a major gap in our understanding of these highly regenerative and cancer-prone tissues. However, recent data identify this missing epithelial stem cell regulator as p63, a member of the p53 family of transcription factors. Disconcertingly, whole-genome ChIp-on-chip data indicate that p63 directly regulates more than 800 genes, a hint that genetic programs underlying self-renewal in stem cells are likely complex and resistant to analysis. In this proposal, we develop the hypothesis that self- renewal is controlled by common mechanisms in stem cells of different lineages, despite the looming contradiction that the known master regulators of self-renewal are tissue-specific. Key to our hypothesis is that these master regulators must coordinate three genetic programs to maintain stem cells, two of which dictate their tissue specificity. Thus in addition to controlling a self-renewal program, these master regulators also need to drive interactions with the stem cell niche as well as forestall tissue-specific differentiation programs. To identify the putative common genetic program underlying self-renewal, we establish experimental strategies in four aims that take advantage of the unique clonogenic properties of epithelial stem cells. In Aim 1, we identify genes involved in self-renewal by "masking" the tissue-specific roles of p63. To do this, we compare the gene expression profiles of phenotypically similar, epithelial stem cells of the same lineage that differ only in their proliferation history. In Aim 2, we identify the genes directly regulated by Bmi-1 in hematopoietic cells and compare these with those regulated by p63 in epithelial stem cells to search for mutually regulated subsets of genes that comprise a common program of self-renewal. In Aim 3, we approach this same question by screening shRNAs against a subset of transcription factors regulated by p63 for their representation over replicative time. Our goal here is to use all three datasets from Aims 1, 2, and 3 to generate a restricted list of candidate genes for individual, in-depth analysis of function in self-renewal pathways. In Aim 4, we examine the molecular mechanisms that control p63 stability as this feature ultimately controls self-renewal. We anticipate that these strategies will provide fundamental insights into the putative "common" pathways by which p63 and other, tissue-specific master regulators impart stemness to regenerative cells. PUBLIC HEALTH RELEVANCE: Answers to the questions posed here will unveil information critical to the therapeutic use of adult stem cells in general and of epithelial stem cells in particular. Given that epithelial tissues contribute to the majority of human cancers, this work should also help to unravel how p63-dependent regenerative properties contribute to the process of tumorigenesis.